Advances in the Properties of Incomptine A: Cytotoxic Activity and Downregulation of Hexokinase II in Breast Cancer Cell Lines

Breast cancer treatments are limited by the cancer subtype and its selectivity towards tumor cells, hence the importance of finding compounds that increase the survival of healthy cells and target any subtype. Incomptine A (IA) is a sesquiterpene lactone with demonstrated cytotoxic activity. In this study, through in vitro assays, it was observed that IA has similar cytotoxic activity between the subtypes triple negative, HER2+, and luminal A of the breast cancer cell lines. IA cytotoxic activity is higher in cancer than in nontumorigenic cells, and its selectivity index for cancer cells is more than that of the drug doxorubicin. Molecular docking and its in silico comparison with the 2-Deoxyglucose inhibitor suggest that IA could bind to Hexokinase II (HKII), decreasing its expression. Since we did not find changes in the expression of the glycolytic pathway, we suppose that IA could affect the antiapoptotic function of HKII in cancer cells. The IA-HKII union would activate the voltage-gated anion channel 1 (VDAC1), resuming apoptosis. Therefore, we suggest that IA could be used against almost any subtype and that its cytotoxic effect could be due to the reactivation of apoptosis in breast cancer cells.


Introduction
Breast cancer is the worldwide most common disease in women since it represents 12% of new cancers in addition to high mortality rates in women [1]. Breast cancer can be classified into different molecular subtypes based on the expression of the receptors that cover the surface of the cancer cells. These proteins are elemental in sending signals for the cells to grow and divide. The different types of breast cancers are identified, then, by the presence or absence of the receptors found on the surface of the cells: the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [2]. The treatment is determined depending on the presence or absence of these receptors; therefore, the strategy will be different depending on the type of cancer in question. Despite the advances in targeted therapies against breast cancer, the drugs used often have the problem that they do not have the same efficacy for any

Cytotoxic Activity of Incomptine A (IA)
The cytotoxic activity of IA in all the cancer cell lines used in this study was evaluated. The 4T1 cell line (triple-negative mouse breast cancer), MDA-MB-231 (triple-negative human breast cancer), SK-BR-3 (overexpressed HER2 breast cancer), T-47D (luminal A human breast cancer), MCF7 (luminal A human breast cancer), and the nontumorigenic MCF10A cell line (epithelial cell line from mammary gland) were used. To compare the cytotoxic effect of IA, we included the dichloromethane extract from the aerial parts of Decachaeta incompta (DEDi), and as positive control, we included the drug doxorubicin (this intersperses into the DNA bases, and through steric obstruction, it inhibits topoisomerase II and the synthesis of DNA or RNA, inducing cell death). Cytotoxicity curves were made from different concentrations of IA (5-18 µg/mL) over 24 h, and the cytotoxic concentration 50 (CC 50 ) was determined for each cell line (Table 1 and Figure 1). To obtain the CC 50 of DEDi and doxorubicin, the concentrations from the cytotoxicity curves were 25-100 µg/mL each (Table 1).  concentration 50 (CC50) was determined for each cell line (Table 1 and Figure 1). To obtain the CC50 of DEDi and doxorubicin, the concentrations from the cytotoxicity curves were 25-100 µg/mL each (Table 1).  The CC50 of IA was much lower compared with DEDi and doxorubicin. The average CC50 for IA in the breast cancer lines, obtained from the values in Table 1, was 6.78 µg /mL, whereas, for DEDi and doxorubicin, it was 50.38 and 57.76 µg/mL, respectively. In the search for new treatments against breast cancer, an important factor is that the drug can be used indistinctly against any subtype. The results show that IA had similar CC50 values in all the breast cancer lines in this study, contrasting the data obtained with the drug doxorubicin, which varied greatly between them, and, even more, affecting MCF10A (the nontumorigenic line). Therefore, IA acts with a lower concentration and more uniformly regardless of the type of breast cancer. On the other hand, a higher concentration (CC50 = The CC 50 of IA was much lower compared with DEDi and doxorubicin. The average CC 50 for IA in the breast cancer lines, obtained from the values in Table 1, was 6.78 µg /mL, whereas, for DEDi and doxorubicin, it was 50.38 and 57.76 µg/mL, respectively. In the search for new treatments against breast cancer, an important factor is that the drug can be used indistinctly against any subtype. The results show that IA had similar CC 50 values in all the breast cancer lines in this study, contrasting the data obtained with the drug doxorubicin, which varied greatly between them, and, even more, affecting MCF10A (the nontumorigenic line). Therefore, IA acts with a lower concentration and more uniformly regardless of the type of breast cancer. On the other hand, a higher concentration (CC 50 = 13 µg/mL) of IA was necessary to carry out cytotoxic activity in the nontumorigenic cells (MCF10A), given that it required more than twice the concentration used in the cancer cells, which shows some specificity towards cancer cells and a lower cytotoxic effect on nontumorigenic cells ( Table 1). The same was observed when IA was compared with DEDi, given that DEDi did not have a better effect than the compound IA. Although it had cytotoxic activity, it was not as uniform as IA, it required a higher concentration in the breast cancer cell lines, and it had greater cytotoxicity towards MCF10A (Table 1).

Specificity of IA for Breast Cancer Cells
As shown in Figure 2, during the IA cytotoxicity assays, in the different cell lines, more morphological changes and a reduction in cell viability in the cancer cell lines were evident in comparation to the nontumorigenic cells. At concentrations of 5-7 µg/mL of IA, between 30-50% of the breast cancer cells, a reduced size, a rounded shape, the loss of cell adherence, and cell death were evident, while, in the nontumorigenic MCF10A line cells, they began to look affected between 10-13 µg /mL. When the cancer cells were treated with 12 µg/mL, practically all of them were found dead; however, in the MCF10A cells, even at 18 µg/mL, 15% of the cells were without alteration, and they seemed viable. As an example, changes in the MDA-MB-231 cancer cells are shown and are compared to MCF10A. Therefore, the specificity of IA towards the breast cancer cells was high, and at a concentration of 7 µg/mL, the effect upon nontumorigenic cells was minimal. Furthermore, its use as an antitumor treatment regardless of the molecular type of cancer promises to be safer ( Figure 2).

Selectivity Index (SI) of IA
To confirm the data observed in the cell culture, the selectivity index (SI) was calculated. The SI indicates the selectivity of a certain compound against cancer cells but not against nontumorigenic cells. The greater the magnitude of the SI, the higher its prefer-

Selectivity Index (SI) of IA
To confirm the data observed in the cell culture, the selectivity index (SI) was calculated. The SI indicates the selectivity of a certain compound against cancer cells but not against nontumorigenic cells. The greater the magnitude of the SI, the higher its preference against tumor cells is [13]. The estimate showed that IA had the highest selectivity towards the cancer cells (range of 1.73-2.32) compared to DEDi (1.3-1.6) and the drug doxorubicin (0.5-1.26) ( Table 2). This supports that IA is a compound with important selectivity towards breast cancer cells that is not affected by the molecular type.

Effect of IA upon Glycolytic Enzymes
To identify the effect of IA upon glycolytic enzymes, modifications in the expression of hexokinase II (HKII), aldolase A (ALDOA), and lactate dehydrogenase (LDH, data not show) were analyzed through Western blotting. Each cell line was treated over 24 h with the CC 50 for IA previously determined. For the cancer cell line 4T1, it was 6.2 µg/mL; for MDA-MB-231, it was 7.6 µg/mL; for SK-BR-3, it was 5.6 µg/mL; for T-47D, it was 7.8 µg/mL; for MCF-7, it was 6.8 µg/mL; and for the nontumorigenic MCF10A, it was 13 µg/mL. The results showed that, in the presence of IA, the expression of HKII decreased significantly after comparing the proportions obtained against the expression of actin in all the cell lines compared to the cells treated with DMSO and the cells without treatment ( Figure 3).

Effect of IA upon Glycolytic Enzymes
To identify the effect of IA upon glycolytic enzymes, modifications in the expression of hexokinase II (HKII), aldolase A (ALDOA), and lactate dehydrogenase (LDH, data not show) were analyzed through Western blotting. Each cell line was treated over 24 h with the CC50 for IA previously determined. For the cancer cell line 4T1, it was 6.2 µg/mL; for MDA-MB-231, it was 7.6 µg/mL; for SK-BR-3, it was 5.6 µg/mL; for T-47D, it was 7.8 µg/mL; for MCF-7, it was 6.8 µg/mL; and for the nontumorigenic MCF10A, it was 13 µg/mL. The results showed that, in the presence of IA, the expression of HKII decreased significantly after comparing the proportions obtained against the expression of actin in all the cell lines compared to the cells treated with DMSO and the cells without treatment ( Figure 3).

Molecular Docking Studies of IA with HKII
To evaluate the existence of putative interaction between IA and HKII, molecular docking was carried out. The docking assays showed that IA bound directly to the HKII

Molecular Docking Studies of IA with HKII
To evaluate the existence of putative interaction between IA and HKII, molecular docking was carried out. The docking assays showed that IA bound directly to the HKII with a binding energy of −6.14 kcal/mol and with polar interactions in the Glu304, Thr336, and Lys337 residues (Table 3 and Figure 4A). To analyze if the binding of IA was sufficient to inhibit the expression of HKII, we compared an already known inhibitor of HKII with 2-Deoxyglucose (2-DG). The 2-DG interaction with HKII presented a binding energy of −3.77 kcal/mol and polar interactions with the Gly299, Glu304, Phe334, and Thr336 residues (Table 3 and Figure 4B). This analysis showed that IA binding to HKII was similar to inhibitor 2-DG binding ( Figure 4C), so IA could be able to bind directly and inhibit HKII like 2-DG does. with a binding energy of −6.14 kcal/mol and with polar interactions in the Glu304, Thr336, and Lys337 residues (Table 3 and Figure 4A). To analyze if the binding of IA was sufficient to inhibit the expression of HKII, we compared an already known inhibitor of HKII with 2-Deoxyglucose (2-DG). The 2-DG interaction with HKII presented a binding energy of −3.77 kcal/mol and polar interactions with the Gly299, Glu304, Phe334, and Thr336 residues (Table 3 and Figure 4B). This analysis showed that IA binding to HKII was similar to inhibitor 2-DG binding ( Figure 4C), so IA could be able to bind directly and inhibit HKII like 2-DG does.

Toxicoinformatic and Pharmaceutical Analysis of IA
A common problem in drug development is that new compounds show remarkable properties in vitro, but when their toxicological and pharmacokinetic properties are

Toxicoinformatic and Pharmaceutical Analysis of IA
A common problem in drug development is that new compounds show remarkable properties in vitro, but when their toxicological and pharmacokinetic properties are reviewed, they are not adequate for clinical use; therefore, it is necessary to perform approaches to the physiochemical and toxicological properties of the compounds and to consider if they have pharmacological potential [13,14]. In this context, we analyzed in silico the physicochemical and toxicological properties of IA to determine if it has pharmacological potential. Using toxicoinformatics tools, we predicted some of these properties for IA (Table 4).

Discussion
Despite the advances in targeted therapies for breast cancer, there is still no treatment that is effective for all subtypes or stages of breast cancer; accordingly, it is necessary to search for new agents with better anticancer activity. In the present study, we demonstrated that the compound incomptine A has cytotoxic activity against breast cancer cells in vitro as well as reduces the expression of the hexokinase II protein. As far as we know, this is the first approximation that suggests that the possible mechanism of action of IA upon tumor cells is through its binding to HKII.
According to the criteria of the National Cancer Institute, a pure compound is considered active when its CC 50 is less than 10 µg/mL [13]. In this case, IA meets the criteria of an active compound for tumor cell treatment since, on average, a concentration of 7 mg/mL is required for a significant CC 50 . In contrast, for nontumorigenic cells (MCF10A) IA has a moderate cytotoxic activity since its CC 50 is 13 mg/mL, and, according to the criteria, if the compound has a CC 50 greater than 10 µg/mL, it is considered moderately active [15]. Therefore, IA could indeed be used as a therapeutic agent, as, at its active concentration (on average, 7 mg/mL), it would not have a significant effect on nontumorigenic cells but would significantly affect breast cancer cells. On the other hand, for doxorubicin to provoke an IA-like effect, a much higher dose is required. Hence, in this way, IA can be considered more efficient. In addition, IA required similar amounts to have a cytotoxic effect on all the tested breast cancer cells regardless of the type of tumor (triple-negative, HER2, or luminal A). This suggests that it could be a compound that acts against any type of breast cancer cell.
Most of the drugs used against breast cancer have a toxic effect on both tumor cells and healthy cells, so, to avoid this toxicity on normal cells, the doses are reduced, which affects the response to treatment of tumor cells. Therefore, it is essential to find compounds with greater selectivity for tumor cells. In this context, IA can be considered to be a bioactive compound and to be nontoxic against normal cells because its selectivity index turned out to be in the range of 1.73 to 2.32 and because it has been reported that an SI greater than 1.0 indicates that a drug is more selective for tumor cells [16]. Meanwhile, doxorubicin was more toxic, and its SI was from 0.5 to 1.23.
Currently, the explored treatment options for breast cancer are focused on drugs that control cell proliferation and induce cell death; however, the side effects are usually severe [17]. Cancer cells enhance their glycolysis, producing lactate even in the presence of oxygen, and the glycolytic enzymes are generally overexpressed, which is an important step for promoting cancer cell survival, proliferation, chemoresistance, and dissemination [18]. Consequently, developing new therapies directed at a different target, such as glycolytic metabolism, would be advantageous to counteract cancer, the reprogramming of its metabolism to a mainly glycolytic one being one of the hallmarks of cancer cells.
HKII, in addition to participating in glycolytic metabolism, is considered an apoptotic regulator since it has physical interactions with the mitochondrial membrane through its binding to VDAC1. HKII associates, through its N-terminal domains, with VDCA1, generating an antiapoptotic event by preventing the release of cytochrome c (Cyt c) [19]. In this context, when we analyzed the effect of incomptine A on the expression of the key enzymes of the glycolytic pathway, such as hexokinase II, aldolase, and lactate dehydrogenase, we found that only HKII showed a significant decrease in its expression and that the others remained stable; this leads us to suggest that the reduction in HKII did not affect the glycolytic pathway and therefore is another affected route in which HKII participates, which could indicate that the cytotoxic effect and mechanism of action of IA are directly related to the other function of HKII.
Molecular docking demonstrated that IA has strong binding with HKII (−6.14 kcal/mol), and, in the in silico comparison with 2-DG (an already described inhibitor for HKII), we observed that both share practically the same binding site, mostly with the amino acid residues GLU304 and THR336, and even that 2-DG has a lower binding strength (−3.77 kcal/mol) than IA, which suggests that the binding of IA to HKII is direct and that this could be the reason for the decrease in the expression of HKII. Additionally, several naturally occurring compounds tested in anticancer studies downregulate HKII with interesting results, where apoptosis is induced. For example, quercetin was shown to inhibit the proliferation of liver cancer cells by decreasing the HKII protein level [20], and Chrysin triggers cell apoptosis in hepatocellular carcinoma cells by targeting HKII [21]. These observations lead us to assume that, like 2-DG, IA binds directly to HKII, causing its inhibition, and, therefore, its binding to VDAC1 does not take place, thus enabling the release of cytochrome c and tumor cell apoptosis. This hypothesis may be supported since it has been reported that, in in vivo models of breast cancer, 2-DG induced oxidative stress and apoptosis [22].
Through computer tools, we performed the prediction of the pharmaceutical and toxicological properties of IA. This analysis showed that IA is a molecule with an adequate pharmacokinetic profile since no mutagenic or tumorigenic effects were observed, and it complies with Lipinski's Rule of Five, which determines if a chemical compound has properties that would make it a medicine that can be used in humans due its pharmacological or biological activity or by assessing drug similarity [23]. On the other hand, in a previous study, the acute oral toxicity of IA was evaluated in a murine model of lymphoma and was comparable with the drug methotrexate, so these predictions can be extrapolated to animals; however, all this needs to be confirmed in animal models of breast cancer and perhaps in future clinical studies [6].
Finally, given that, in cancer, the overexpression of HKII and its association with VDAC1 in the mitochondrial membrane of cancer cells inhibits apoptosis, we propose that, in breast cancer cells, the cytotoxic activity of IA is the consequence of its binding to HKII. IA-HKII binding provokes the release of VDAC1, which, in turn, will allow the exit of Cyt c, and apoptosis will occur ( Figure 5). This first approximation will give us the guidelines to carry out more specific analyses and corroborate this hypothesis in in vivo models.
icological properties of IA. This analysis showed that IA is a molecule with an adequate pharmacokinetic profile since no mutagenic or tumorigenic effects were observed, and it complies with Lipinski s Rule of Five, which determines if a chemical compound has properties that would make it a medicine that can be used in humans due its pharmacological or biological activity or by assessing drug similarity [23]. On the other hand, in a previous study, the acute oral toxicity of IA was evaluated in a murine model of lymphoma and was comparable with the drug methotrexate, so these predictions can be extrapolated to animals; however, all this needs to be confirmed in animal models of breast cancer and perhaps in future clinical studies [6].
Finally, given that, in cancer, the overexpression of HKII and its association with VDAC1 in the mitochondrial membrane of cancer cells inhibits apoptosis, we propose that, in breast cancer cells, the cytotoxic activity of IA is the consequence of its binding to HKII. IA-HKII binding provokes the release of VDAC1, which, in turn, will allow the exit of Cyt c, and apoptosis will occur ( Figure 5). This first approximation will give us the guidelines to carry out more specific analyses and corroborate this hypothesis in in vivo models. Figure 5. Proposed mechanism of action for IA in breast cancer cells. In breast cancer, KHII is overexpressed and bound to VDCA1, which prevents the presence of apoptosis. When cells are treated with IA, it binds to HKII, preventing the binding of HKII with VDAC1 and leaving the pore free with which apoptosis can be initiated.  Proposed mechanism of action for IA in breast cancer cells. In breast cancer, KHII is overexpressed and bound to VDCA1, which prevents the presence of apoptosis. When cells are treated with IA, it binds to HKII, preventing the binding of HKII with VDAC1 and leaving the pore free with which apoptosis can be initiated.

Incomptine A
DEDi and IA were donated by Dr. Fernando Calzada, which were isolated according to the method described by Calzada et al., 2009 [24]. The identification of incomptine A was made through nuclear magnetic resonance with an authentic sample with a purity of 99%.

In Vitro Cytotoxicity Assay
Cell cytotoxicity was measured using the WST-1 assay, which is based on the reduction of tetrazolium salt, WST-1, to formazan by cellular mitochondrial dehydrogenase [25].

Cell Morphology Analysis
Morphological alterations and cell damage were qualitatively investigated using an inverted phase contrast fluorescence microscope (Olympus CKX41), and the photos were taken with a digital camera.

Western Blotting
After the treatment of DEDi and IA, breast cancer and 10A cells were lysed with RIPA lysis buffer containing protease cocktail (Santa Cruz Biotechnology, Dallas, TX, USA) on ice for 15 min. The cell lysate was harvested and centrifugated at 10,000 × g for 10 min, and the supernatant was collected. The protein concentrations were determined with Quick Start™ Bradford Protein Assay Kit 1 (5000201). Thirty micrograms per sample were subjected to SDS-PAGE and were then transferred onto polyvinylidene difluoride membranes (Merck IPVH00010). After blocking the nonspecific binding site on the membrane with 5% nonfat milk solution, they were incubated with the following protein-specific antibodies overnight at 4 • C: mouse anti-HKII (1:2500, Santa Cruz Biotechnology, sc-130358), mouse anti-ALDOA (1:2500, Santa Cruz Biotechnology, sc-390733), and mouse anti-LDH (1:2500, Santa Cruz Biotechnology, sc-133123). Donkey antimouse antibody conjugated to horseradish peroxidase (HRP) (1:2500, abcam, ab6820) was used for secondary detection, and samples were visualized with chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Molecular Docking Studies
The chemical structures of incomptine A (IA) (CID: 118707242) and 2-deoxyglucose (2-DG) (CID: 108223) were retrieved from the chemical library PubChem (https://pubchem. ncbi.nlm.nih.gov/) (accessed on 20 July 2023). These were optimized and submitted to energetic and geometrical minimization using the Avogadro software. The 3D structures involved in the glycolytic metabolism of cancer cells were retrieved from the Protein Data Bank (https://www.rcsb.org) (accessed on 20 July 2023) with the following accession codes: hexokinase II (PDB ID: 2NZT). The total molecules of water and ions that were not needed for catalytic activity were stripped to preserve the entire protein. All polar hydrogen atoms were added and ionized in a basic environment (pH = 7.4), and Gasteiger charges were assigned. The computed output topologies from the previous steps were used as input files for docking simulations. The molecular docking experiments were carried out using AutoDock 4.2 software. The grid was centered at the following coordinates: HXII (center x = −3.228, center y = 13.415, and center z = 16.595) with grid dimensions of 60 × 60 × 60 points. The Lamarckian genetic algorithm was employed as a scoring function with a randomized initial population of 100 individuals and a maximum number of energy evaluations of 1 × 107 cycles. The analysis of the interactions in the enzyme/inhibitor complex was visualized with PyMOL software (the PyMOL Molecular Graphics System, Ver 2.0, Schrödinger, LLC, NY, USA). The validation of molecular docking was carried out by redocking the cocrystallized ligand in the receptors. The lowest energy pose of the cocrystallized ligands was superimposed, and it was observed whether it maintained the same binding position. The RMSD were calculated, and a reliable range within 2 Å was reported.

Docking Validation Protocol
The validation of the molecular docking results was conducted by redocking the cocrystallized ligand in the receptor (PDB ID: 2NZT). The lowest energy pose of redocking and the cocrystallized ligands were superimposed, and it was observed whether it gained the same position; additionally, its RMSD was calculated between these two superimposed ligands. The RMSD was within a reliable range of 2 Å.

In Silico Toxicology and Pharmaceutical Properties
There are different free access programs that are used to determine if a molecule possesses some pharmacological potential and possible toxicological risks through human consumption. The two-dimensional and three-dimensional structures of incomptine A were obtained from Pubchem CID 118707242. The simplified line entry system of molecular entry (SMILES) of incomptine A was used in the free access software Molinspiration, SwissAdme, and ADMETsar to evaluate the physicochemical properties.

Statistical Analysis
The results are expressed as mean ± standard error of the mean (SEM). Statistical analysis of the data was performed using one-way ANOVA with a value of p < 0.05 to establish a significant difference between the study groups. The CC50 was calculated through linear interpolation of the percentage mortality values for each concentration. All analyses were performed using Graph Pad Prism version 8 (GraphPad Software Inc., La Jolla, CA, USA).

Conclusions
IA can be considered to be more efficient in its cytotoxic activity and selectivity for breast cancer cells, and it can act in a similar way regardless of the cancer subtype (triplenegative, HER2, or luminal A); therefore, it is a compound that could be used against almost any subtype of breast cancer. According to the prediction of Lipinski's Rule of Five, the physicochemical and toxicological properties of IA show that it is a molecule with an adequate pharmacokinetic profile, that it would not present mutagenic or tumorigenic effects, and that it could potentially be used in in vivo studies and in the future in clinical studies.
We suggest that the mechanism by which IA could be leading to the death of tumor cells is through its binding to HKII, which, in turn, no longer joins to VDAC1, thus allowing the resumption of apoptosis. However, more studies are needed to confirm this hypothesis.